PROJECT 3 ABSTRACT Age-associated thymic involution plays a critical role in the deterioration of T cell-mediated immunity, thereby contributing to the increased susceptibility of older individuals to infectious disease, cancer, and autoimmunity. The involuted thymus produces fewer nave T cells impairing the generation of protective immune responses against newly encountered antigens; moreover, defects in the aged thymic environment may lead to inefficient central tolerance induction, potentially contributing to the increased incidence of autoimmunity with age. Thymocyte maturation depends on indispensable survival, differentiation, and proliferative cues provided by heterogeneous thymic stromal cells. Deterioration of the thymic stromal compartment is largely responsible for age-associated thymic involution. However, the underlying cellular and molecular mechanisms resulting in stromal decline and the precise impact on T cell development and selection remain unknown. The premise of Project 3 is that declining function of aging thymic stromal cells is due to altered gene expression that impairs multiple stages of T cell differentiation. We hypothesize that identifying specific molecular and cellular age-associated defects in thymic stromal cells will provide a basis for devising strategies to restore thymic function to maintain a healthy T cell compartment throughout the lifespan. In Aim 1, we will determine if the loss of early thymocyte progenitors (ETP) during thymic involution is due to an age- associated decline in the quantity and/or quality of ETP niches, and whether effective rebound therapies restore the ETP niche. In Aim 2, we will test the hypothesis that as the thymic epithelial cell (TEC) compartment deteriorates, thymocytes are no longer efficiently recruited into the medulla, and negative selection is impaired, which could allow escape of autoreactive T cells. In Aim 3, we will perform transcriptional profiling to identify age-regulated genes expressed in TEC that regulate TEC homeostasis and support of thymopoiesis. Subsequent functional analysis will determine the impact of candidate genes on thymic function. P3 has multiple points of intersection with all other Projects and Cores. For example, we will provide data on molecular signatures of aging mouse TEC and endothelial cells (EC) and corresponding human thymic data (Core C) to the Human-Mouse Timeline (P1). For analysis of ETP niche quantity, we will collaborate with Core B, which will also provide general Biostatistics support throughout. Analysis of age-associated changes in TEC and EC will be integrated (through Core A) with data from P2 on changes in EC that impact thymic rebound. In addition, we will compare molecular and cellular drivers of thymic involution with aging-associated changes identified in lymph nodes (P4), to facilitate design of novel strategies to rejuvenate both central and peripheral lymphoid organs (Core D). Thus, the results generated in P3 are integral to achieving the overall Program goals of elucidating mechanisms responsible for thymic and peripheral immunosenescence, and devising and evaluating rational therapeutic strategies to rejuvenate the T cell compartment.